滋养层糖蛋白，即所谓的 5T4，是一种在许多不同癌症中表达的癌胎儿抗原。然而，临床上并没有采用5T4特异性放射性配体进行无创诊断。因此，本研究的目的是开发一种用于临床前和临床阶段5T4表达成像的PET放射性示踪剂。通过骆驼免疫构建VHH文库。通过噬菌体展示生物淘选和周质提取物酶联免疫吸附测定筛选 VHH 对 5T4 抗原的特异性。将 1,4,7-三氮杂环壬烷-1,4,7-三乙酸 (NOTA) 衍生物与选定的 VHH 缀合。放射性标记后，使用 BxPC-3 和 MDA-MB-468 荷瘤小鼠进行 microPET/CT 和离体生物分布。将冷 VHH 与示踪剂共同注射以挑战其在体内的结合。在初步临床研究中，对经病理证实的原发性和转移性肿瘤的患者注射示踪剂后1小时采集PET/CT图像。成功免疫骆驼后，构建了容量为1.2 × 1012集落形成单位的文库。选择中值效应浓度为0.43 nM的Nb1-40。人源化后，所得的 H006 保持了对 5T4 的高亲和力。制备的[68Ga]Ga-NOTA-H006的摩尔活度为6.48-54.2GBq/μmol，具有高放射化学纯度(>98%)。使用 [68Ga]Ga-NOTA-H006，microPET/CT 清晰地显示了 BxPC-3 荷瘤小鼠中 5T4 的表达。离体生物分布表明，注射后 60 分钟时达到最高的肿瘤与血液比率（~ 3 倍）和肿瘤与肌肉比率（~ 5 倍）。以 1.5 mg/kg 的剂量共同注射冷 H006 显着降低了肿瘤摄取 (p<<0.0001)。在初步临床研究中，[68Ga]Ga-NOTA-H006 证明了其绘制人类 5T4 阳性病变图谱的能力，并产生了 3.4 × 10- 2 mSv/MBq 的平均有效剂量。[68Ga]Ga-NOTA-H006，已成功开发出可在体内可视化 5T4 表达的技术。这为通过核医学非侵入性研究 5T4 表达提供了机会。有必要进行进一步的临床研究，以探索其在疾病进展和伴随诊断中的临床价值。© 2024。作者获得 Springer-Verlag GmbH 德国（Springer Nature 旗下公司）的独家许可。

Trophoblast glycoprotein, the so-called 5T4, is an oncofetal antigen expressed in many different cancers. However, no 5T4-specific radioligand is employed in the clinic for non-invasive diagnosis. Thus, the aim of the current study was to develop a PET radiotracer for imaging 5T4 expression in preclinical and clinical stages.A VHH library was constructed by camel immunization. The specificity of the VHHs toward 5T4 antigen was screened through phage display biopanning and periplasmic extract enzyme-linked immunosorbent assay. 1,4,7-Triazacyclononane-1,4,7-triacetate acid (NOTA) derivative was conjugated to the selected VHH. After radiolabeling, microPET/CT and ex vivo biodistribution were conducted using BxPC-3 and MDA-MB-468 tumor-bearing mice. Cold VHH was co-injected with the tracer to challenge its binding in vivo. For the pilot clinical study, PET/CT images were acquired at 1 h after injection of tracer in patients with pathologically confirmed primary and metastatic tumors.A library with a capacity of 1.2 × 1012 colony-forming units was constructed after successful camel immunization. Nb1-40 with a median effect concentration of 0.43 nM was selected. After humanization, the resulting H006 maintained a high affinity towards 5T4. [68Ga]Ga-NOTA-H006 with the molar activities of 6.48-54.2 GBq/µmol was prepared with high radiochemical purity (> 98%). Using [68Ga]Ga-NOTA-H006, microPET/CT revealed a clear visualization of 5T4 expression in BxPC-3 tumor-bearing mice. Ex vivo biodistribution showed that the highest tumor-to-blood ratio (∼ 3-fold) and tumor-to-muscle ratio (∼ 5-fold) were achieved at 60 min post-injection. Co-injection of the cold H006 at a dose of 1.5 mg/kg significantly reduced the tumor uptake (p < 0.0001). In the pilot clinical study, [68Ga]Ga-NOTA-H006 demonstrated its capacity to map 5T4-positive lesions in humans and yielded a mean effective dose of 3.4 × 10- 2 mSv/MBq.[68Ga]Ga-NOTA-H006, which can visualize 5T4 expression in vivo, has been successfully developed. This opens up opportunities for non-invasively studying 5T4 expression through nuclear medicine. Further clinical investigations are warranted to explore its clinical value in disease progression and companion diagnosis.© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.